Recent biophysical studies have made great progress in clarifying the function of single molecular motors in vitro. However, in vivo molecular function is far more complicated than these in vitro studies predict: contrary to the uni-directional motion observed in vitro, many vesicles or other cargos move bidirectionally along microtubules, frequently reversing their direction of travel. Nonetheless, transport can be regulated, so that the cargo moves-on average-to the right place. To understand how such transport is regulated requires the development of new tools capable of quantifying-in vivo-the motion of individual cargos with high temporal and spatial resolution. The research develops such biophysical tools, and combines them with genetics and biochemistry to investigate the function of motors or motor complexes in vivo. Specifically, bi-directional motion of lipid droplets in early Drosophila embryos is investigated. The work can be conceptually divided into two complimentary approaches. The first, the biophysical characterization of the effects of mutations, uses optical tweezers and particle tracking and analysis to determine the specific physical role of proteins in the transport pathway. One goal is to test a theoretical framework hypothesizing the existence of a complex with certain functions, and place each protein within this framework. The second approach employs biochemical techniques to identify additional proteins involved in the regulation of transport, and determine relevant interactions between the different proteins. This information will clarify at the molecular level how the biophysically determined functions come about, and will directly investigate the hypothesized complex. The function of the proteins identified in the biochemical approaches will be directly investigated using the biophysical assays. Bi-directional transport is directly related to public health: viruses such as herpes spread through cells in a bi-directional manner; many important cargos like mitochondria and endosomes move bi-directionally. Further, mutation in some of the genes investigated here, such as Lisi, lead to human birth defects. Finally, a better understanding of transport might allow the design of new drug delivery systems.